Crohn's disease (CD) is a potentially debilitating, chronic inflammatory disorder of the gastrointestinal tract. Moreover, current medications for this disorder tend to be broad-spectrum immunosuppressive agents with the potential for profound side effects. Thus there is a strong rationale to develop less toxic therapeutic options for CD, particularly in pediatric patients who may receive decades of potent immunosuppressive therapy. Although the precise etiology of CD remains unclear, altered epithelial function plays a key role in its pathogenesis. Therefore, the long-term goal of this research is to develop safer therapies for CD that augment intestinal epithelial function, as opposed to suppressing the immune system. Intestinal stem cells (ISCs) located at the base of intestinal crypts play a central role governing proliferation and differentiation of the gut epithelium. Although host pathways that regulate ISC function are progressively being elucidated, the effects of exogenous factors on ISC biology are poorly understood. In particular, the precise influences of the commensal enteric microbiota on ISC biology have not been characterized. Hence, the objective of the current proposal is to elucidate how the gut microbiota modulates ISC function. This objective will be achieved by testing the central hypothesis of this study, namely that the intestinal microbiota is a key regulator of ISC maintenance and proliferation. This hypothesis has been formulated based upon supporting preliminary data generated by the applicant, and will be tested via two specific aims. First, the role of the intestinal microbiota in regulating IS biology will be examined in healthy mice. This will be accomplished using established flow cytometry approaches, as well as complementary in vitro and in vivo techniques to compare ISC function in conventional (CV) versus germ-free (GF) mice. Second, the role of Paneth cells (PCs) as critical mediators of microbial-ISC interactions will be explored. This aim is built upon the rationale that PCs are a key component of the ISC niche, and are able to directly sense commensal gut bacteria. To accomplish this aim, established techniques for isolating PCs and ISCs will be used to assess the ability of GF versus CV PCs to support ISC growth in vitro. These data will be enhanced by in vivo characterization of ISC function in Irgm1-/- mice, which have clear PC abnormalities. The proposed approach is innovative because it focuses on a novel regulatory role for the gut microbiota, namely its ability to modulate PC niche signaling to the ISC compartment. This is in contrast to previous work that has concentrated on the role of the microbiota as a regulator of PC antimicrobial function. This research is significant because it is expected to lead to the development of microbial modulation strategies that will allow for the manipulation of ISC function for therapeutic purposes. Ultimately, this has the potential to minimize immunotoxic therapies for patients with CD, while concomitantly promoting mucosal healing. Importantly, the data and techniques established through this proposal will form the foundation for further independent studies that define the mechanisms by which intestinal bacteria regulate ISC biology.